- Email: [email protected]
Enhanced activity of the plasma membrane localized callose synthase in cucumber leaves with induced resistance
Physiological and Molecular Plant Pathology (1990) 37, 221-228
221
Enhanced activity of the plasma membrane localized callose synthase in cucumber leaves with induced resistance 1 . SCHMELE and H. KAUSSt Fachbereich Biologie der Universität Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, F .R.G . (Accepted for publication July 1990)
The resistance of the first true leaf of cucumber to Colletotrichum lagenarium was increased by infecting the cotyledon or the tip of the first leaf with C . lagenarium or tobacco necrosis virus . Microsomes and plasma membranes, prepared from first leaves in which resistance was induced, exhibited increased specific activity of the Cat'-regulated 1,3-ß-glucan (callose) synthase . This enzyme activity is latent in epidermal cells of unchallenged healthy leaves but after activation renders the cells capable of a rapid production of callose-containing papillae . Activation occurs as soon as the Ca" permeability of the plasma membrane is perturbed by attempted fungal penetration . INTRODUCTION Systemically induced resistance in cucurbits against Colletotrichum lagenarium is associated with an increased ability of the host to prevent penetration of the epidermal cell wall [9, 13, 24] . This induced penetration might result, in part, from the ability of the cells to react by cell wall lignification at the site of attempted penetration [9] . The increased lignification in systemically resistant plants is reflected in cucumber at a biochemical level by enhanced peroxidase activity [10] and in muskmelon by increased hydroxycinnamate : CoA ligase activity [T] . It appears that induced resistance involves a developmentally regulated increased synthesis of enzymes that allows defence reactions to proceed more effectively when required . The systemic induction of chitinase [21] and 1,3-ß-glucanase [2] in cucumber and of chitinase in muskmelon [22] also involves enzyme synthesis . These lytic enzymes could operate by directly suppressing the growth of fungal hyphae in the course of penetration [20] . Consistent with this idea is the localization of chitinase in the apoplastic space of cucumber [3] . As an additional defence-mechanism, callose-containing papillae [2] are formed earlier and are more efficient in preventing penetration in cucumber leaves exhibiting induced resistance [24] . The activity of the plasma membrane-localized Ca"-dependent 1,3-ß-glucan synthase is latent in suspension cells but increases rapidly when local perturbations of the plasma membrane result in an influx of Ca" [15-17, 25] . The research reported in this paper shows that the activity of 1,3-,O-glucan f To whom correspondence should be addressed . 0885--5765/90/090221 +08 $03 .00/0
© 1990 Academic Press Limited I3-2
222
I . Schmele and H . Kauss
synthase is systemically enhanced in resistant' leaves and this enzyme might enable the plasma membrane to form papillae by more `rapidly depositing callose .
MATERIAL AND METHODS
Plant materials Cucumber plants (Cucumis sativus L . cv . Mervita) were grown as described [23] . Induction of systemic resistance was performed by placing six drops (10 µl) of a spore suspension (10 6 spores ml - ') of C. lagenarium on one cotyledon of plants in which the first true leaf had nearly expanded . Alternatively, both cotyledons were gently rubbed with a suspension of tobacco necrosis virus (TNV) containing 6 mg ml -1 Celite [23] . The plants were then allowed to grow for various times before systemic resistance was assessed by placing 20 drops (5 µl) of a C . lagenarium spore suspension (10 6 spores ml - ') on the first leaves .
Microsome preparations The first true leaves from four to seven plants (as indicated) were cut, and 0 . 5 g were homogenized at 0 °C [5, 23] in 5 ml of 100 mm Hepes/NaOH (pH 7 . 0) containing 10 mm EGTA and 5 mm dithiothreitol . The homogenate was centrifuged at 480 g for 5 min and the pellet (cell wall debris plus some adhering parts of plasma membranes) then resuspended in 5 ml of 50 mm Hepes/NaOH (pH 7 . 0) containing 1 mm dithiothreitol. Microsomes from the supernatant were pelleted at 48000 g for 5 min and the pellet washed twice in 5 ml of 50 mm Hepes/NaOH (pH 7 . 0) containing 1 mm dithiothreitol before resuspending in 5 ml of the same buffer .
Plasma membrane preparations Leaves (2 . 5 g) were homogenized in 10 ml of 100 mm Hepes/NaOH (pH 7 . 0) containing 5 mm dithiothreitol, 0.25 M sucrose and 10 mm EDTA . The homogenate was fractionated by centrifugation as described [5] and the microsome fraction suspended in 3 ml of 10 mm Hepes/NaOH (pH 7 . 0) containing 0 . 25 M sucrose and 1 mm EDTA . The microsome suspension (1 . 24 g, about 1 ml) was added to the same buffer (8. 15 g) containing 0 . 58 g polyethyleneglycol 4000 and 0 . 58 g dextran T500 after cooling to 0 ° C and mixed gently [19] . To allow separation of the total 3 ml of microsome fraction, three samples were run in parallel . They were centrifuged (1000 g for 3 min at 2 °C) and about 90% of each upper phase was recovered . A fresh upper phase was prepared by centrifugation of the same mixture as above but without microsomes, and this was added to each lower phase to give a second mixture and recentrifuged . The combined upper phases from each of the three samples were diluted 5-fold by volume with 10 mm Hepes/NaOH (pH 7 . 0) containing 1 mm EDTA and the membranes pelleted by centrifugation (100000 g, 1 h) . The resulting plasma membrane fraction was resuspended in I ml of 50 mm Hepes/NaOH (pH 7 . 0) containing 1 mm dithiothreitol .
Assay of 1,3-#-glucan synthase The enzyme assay [5] used 50 µl of the membrane suspension and 50 µi of assay buffer . In some experiments (Tables 2 and 3) the assay buffer consisted of 50 mm Tes/NaOH
223
Activity of callose synthase in cucumber TABLE I
Activity of the 1,3-ß-glucan synthase in various subcellular fractions derived from the first leaves of control and systemically resistant cucumber plants
Activity' in first leaves from Control Crude homogenate' After centrifugation at 480 g Supernatant Pellet Washed microsomes' Specific activity of microsomes' Specific activity [%] Lesions' Number Diameter [mm]
Resistant
53
63
61 15 30 11 100
76 19 43 18 163 3-5+0-7 1 . 6±0. 5
19-0+1-4 4. 6±0 . 1
'Total mU in the preparation derived from 0 . 5 g of leaves . °A single cotyledon on each of seven seedlings was inoculated with C. lagenarium and 7 days later the first leaves were harvested and 0 . 5 g homogenized in 5 ml of butler. Control plants were not inoculated . `Microsomes were twice pelleted and resuspended in buffer (5 ml per 0. 5 g of leaves) . '[mU mg- ' protein] . 'Resistance was assayed by challenge inoculating with 20 drops of C . lagenarium spores on the first leaves of two plants grown in parallel .
TABLE
2
Specific activity of microsomal 1,3-fl-glucan synthase in cucumber leaves infected at the tip with TNV to induce systemic resistance against C . lagenarium
Specific activity [mU mg - ' protein] Segment
Control
Resistant'
Increase [-fold]
1 (tip)° 2 3 4 (base)
5. 2 5.3 3.2 3.7
22. 0 15 . 1 16. 4 17. 0
4 .2 2.8 5.1 4 .6
'Induced systemic resistance was assayed by spreading 20 drops of C. lagenarium spores over the entire first leaf surface except for the tip, 4 days after inoculating the tip with TNV . 'The leaves from 4 plants were infected at their tip (about 1 . 5 cm) with TNV and cut after 4 days into four segments of equal width . The segments were homogenized and microsomes prepared as described in footnotes ° and a in Table 1 .
(pH 7 . 0) 16 % w/v glycerol, 20 mm cellobiose, 0 .04 % w/v digitonin, 10 mm MgCl,, 4 mm EGTA, 4 mm CaCl2' It contained a final free Ca" concentration of about 60 RM . In other experiments (Table 1) it consisted of 50 mm Hepes/NaOH (pH 7 . 0) with the same additions . As certain cell fractions assayed with this buffer contained some EDTA, the final concentration of free Ca" was between 0 . 1 and 1 . 0 mm, thus providing conditions in which the enzyme was saturated with Ca" [5, 15, 18] . The enzyme
224
I . Schmele and H . Kauss TABLE 3
Specific activity of 1,3-ß-glucan synthase in plasma membranes from the first leaves of control and systemically resistant cucumber plants
First leaves from Control Microsomes Total activity [mU]' Spec . activity [mU mg r protein] Plasma membranes° Total activity [mU] ° Spec . activity [mU mg' protein] Purification [-fold] Lesions' Number Diameter [mm]
30 .3 10 11 .7 140±29 14 18. 7±0. 5 5 . 1±0 . 5
Resistant'
78 .6 21 (2. 1)e 30 .4 293+21 (2. 1)c 13 .9 4 .0±1 . 6 1 .6±0. 4
'Systemic resistance was induced as described in Table 1 . 'Total activity in the preparation derived from 2 . 5 g leaf fresh weight . 'Increase over control [-fold] . ° Plasma membranes were prepared from microsomes in three PEG/dextran two phase systems run in parallel . The means ± SD of the specific activities from these three samples are given . 'Resistance was assayed by challenge inoculating the first leaves with 20 drops of C . lagenarium spores . The results are means+ SD on the first leaves from three plants .
reaction was started by the addition of 5 gl of radiolabelled UDP-glucose to give a final concentration 0.8 mm [5] . The reaction was terminated after 10 or 20 min at 25 ° C and the amount of product determined [5] . Protein was measured with Coumassie blue [5] using BSA as a standard . RESULTS 1,3-ß-glucan
synthase
activity in microsomes
All subcellular fractions from the true first leaves of plants exhibiting systemic resistance induced by C. lagenarium showed an increased 1,3-ß-glucan synthase activity over the uninduced controls (Table 1) . However, in both the control and resistant plants the total enzyme activity in the supernatants was higher than in the crude homogenates, although the resuspended pellets also exhibited some activity because of plasma membrane remains adhering to cell wall debris . The higher 1,3-ß-glucan synthase activity in the supernatants is most likely due to the fact that a significant fraction (about one third) of the crude homogenate volume is composed of bulky leaf constituents devoid of activity (e .g. cell walls and nuclei) and their removal as a pellet increases the proportion of microsomes in the samples assayed from the supernatants . The increased 1,3-#-glucan synthase activity was shown to be membrane-associated by measurement of the specific activity of the washed microsomes (Table 1) . In five repeat experiments similar to the one described in Table 1, the specific activity of 1,3ß-glucan synthase in the microsomes from resistant leaves was 157 ± 22 % of that in the control plants .
Activity of callose synthase in cucumber
225
The increase in specific activity was not specific to plants induced by C . lagenarium since in five similar experiments using TNV as the inducing agent, an increase in the specific activity of 1,3-fl-glucan synthase of 184±38% was observed 4 days after inoculation . However, no increase in 1,3-fl-glucan synthase was observed in the first leaves 7 days after inoculation of the cotyledons with TMV (data not shown), a pathogen that does not produce necrotic lesions nor induce systemic resistance in cucumber [13] . Inoculating the tip of the first true leaf with TNV induced systemic resistance in the rest of this leaf against C. lagenarium . The results of one experiment, given in Table 2, show that 17-5+2-3 lesions developed per leaf in the control uninduced plants while only 5 . 5 ± 2 . 1 lesions developed in TNV-induced leaves 7 days after challenge infection . The lesions on the control plants were also larger than on the induced resistant plants . The specific activity of 1,3-ß-glucan synthase in microsomes from an area of the leaf adjacent to the site of TNV-inoculation as well as from the entire leaf was considerably higher 4 days after inoculation than in controls (Table 2) . In eight similar repeat experiments the increase in 1,3-fl-glucan synthase at the tip was 2 . 6±0 . 7 fold greater than in the controls . A time course study revealed that the earliest detectable increase in 1,3-ß-glucan synthase occurred in the tip segments 2 days after inoculation and was fully expressed throughout the leaves 2 days later (data not shown) . 1,3-fl-glucan synthase activity in the plasma membrane
Partitioning the microsomes in a two phase PEG/dextran system, which selects for sealed rightside-out plasma membrane vesicles [5, 19], resulted in a similar enrichment of the 1,3-fl-glucan synthase activity in control and resistant plants with a two-fold higher specific activity in the plasma membranes from systemically resistant plants (Table 3) . Five repeat experiments showed a mean increase in the specific activity in the plasma membranes from resistant leaves of 183±25% . Some properties of 1,3-ß-glucan synthase from cucumber
1,3-fl-glucan synthase activity in microsomes from cucumber leaves was studied with regard to certain regulatory properties . The detailed results are not given because the properties were in general similar to those reported for other plant species . Thus results are presented here as a summary . The cucumber enzyme requires Ca" for activity, with a half-saturation below 1 µM free Ca", even in the presence of 5 mm Mg" . In the absence of Mg", Ca" activation was synergistically enhanced by 50 IAM spermine and additively by poly-L-ornithine (25 µg 125 .tl - ' assay) . Enzyme activity under standard assay conditions increased up to 10-fold after addition of digitonin (0 . 019 %, w/v, final conc .), presumably due to the fact that the 1,3-ß-glucan synthase was present in rightside-out plasma membrane vesicles and the digitonin increased the permeability of the vesicles and thus increased access for substrate and activators to the cytoplasmic membrane surface . The substrate saturation curve was sigmoidal between 10 and 100 gm UDP-glucose, indicating a complex regulatory behaviour . Therefore, about 0 . 8 mm UDP-glucose was required for the standard assay . The properties of the enzyme in microsome fractions of control and sytemically resistant leaves were similar, but the enzyme from induced resistant leaves exhibited higher activities at the same activator concentrations .
226
I . Schmele and H . Kauss
DISCUSSION The systemically induced resistance in cucumber to fungi is clearly associated with a decrease in penetration efficiency [9, 24, 26] . This reduced efficiency appears to result from a number of factors which result from mechanisms that are systemically programed during the pre-infection induction process and then become triggered by the fungal attack . Some fungal penetration attempts fail without any cytologically visible host reaction [24] . The presence of preformed fungitoxic substances such as chitinase [3] in the apoplast of induced plants may relate to this observation . Cytologically detectable reactions are involved including the incorporation of ligninlike material in the epidermal cell wall [9] and/or the deposition of papillae below the area of attempted penetration [24, 26] . The ability to react rapidly in this manner appears to result from a developmentally regulated induction of enhanced activities of enzymes, as previously shown for enzymes involved in phenylpropanoid production [7] and polymerization [10] . Papillae, in general, have been shown to contain callose among other substances [ref. 1, 8, 12, and literature cited there] . These cell wall appositions occur at very localized sites and their composition can only be evaluated by relatively non-specific histochemical methods . The contribution of callose deposition in papillae to resistance remains, therefore, controversial . Recently, careful time-course studies combined with the use of 2-deoxy-n-glucose to inhibit callose formation in the ml-o barley/powdery mildew system have provided evidence that callose deposition can contribute to resistance [1] . In cucumber, rapid formation of full-size papillae is associated with penetration inhibition in systemically resistant plants [2, 24] . Cucumber papillae are poorly characterized biochemically although electron-dense and -opaque layers have been observed in electron micrographs [26], possibly corresponding to lignin-like and polysaccharide material, respectively . The presence of callose in cucumber papillae has been shown by fluorescence with aniline blue [2] . The properties of the cucumber 1,3-fl-glucan synthae, namely its stimulation by Ca", Mg", digitonin, spermine or poly-t .-ornithine as well as its partitioning in the two phase system (Table 3), all show that a plasma membrane-located 1,3-#-glucan synthease, formerly called glucan synthase II . This enzyme has been implicated in callose synthesis [5, 11, 14-16, 18] . Several unsuccessful attempts to purify this enzyme have been made [4, 6] . However, these attempts have provided indirect evidence that the enzyme has a complex structure and probably consists of several subunits exhibiting different functions . Thus, no conclusions about whether the increased specific activity measured under standard assay conditions in microsomes and plasma membranes from systemically resistant leaves is due to an overall increase in all enzyme components or only relate to the regulatory Ca"-binding subunits can be drawn . The observation that the 1,3-#-glucan synthase activity in the plasma membranes of cucumber leaves with systemically induced resistance is enhanced, provides further evidence for the general notion that defence-related enzymes become more active due to long-term developmental processes . In the case of callose formation, allosteric Ca" activation is a major parameter of short-term regulation of 1,3-#-glucan synthase, although additional signals are likely to be required [16, 17] . This activation mechanism
227 Activity of callose synthase in cucumber for latent 1,3-#-glucan synthase, which occurs with higher activity in cucumber leaves exhibiting increased resistance, is consistent with the cytological observation that in these leaves a higher proportion of full-sized papillae is formed below the site of fungal attack [24] . How callose formation co-operates with the other processes which contribute to papillae formation remains to be established . This work was financially supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie .
REFERENCES 1 . BAYLES, C . J ., GHEMAWAT, M . S., AIST, J . R . (1990) . Inhibition by 2-deoxy-D-glucose of callose formation, papillae deposition, and resistance to powdery mildew in an ml-o barley mutant . Physiological and Molecular Plant Pathology 36, 63-72 . 2 . BINDER, A., BAER, G ., HOFMANN, C. & KOVATS, K . (1989) . Mechanisms in systemic induced disease resistance. In Signal Molecules in Plants and Plant-Microbe Interactions, Ed . by B, J . J . Lugtenberg, pp . 267-272 . Springer Verlag, Berlin . 3 . BOLLER, Ta. & MÉTRAUX, J . P . (1988) . Extracellular localization of chitinase in cucumber . Physiological and Molecular Plant Pathology 33, 11-16 . 4 . DELMER, D . P . (1987) . Cellulose biosynthesis . Annual Review of Plant Physiology 38, 259-290 . 5 . FINK, J ., JEBLICK, W ., BLASCHEK, W . & KAUSS, H . (1987) . Calcium ions and polyamines activate the plasma membrane-located 1,3-#-glucan synthase. Planta 171, 130-135 . 6 . FINK, J ., JEBLICK, W. & KAUSS, H . (1990) . Partial purification and immunological characterization of 1,3-fl-glucan synthase from suspension cells of Glycine max. Planta 181, 343-348 . 7 . GRAND, C . & ROSSIGNOL, M . (1982/83) . Changes in the lignification process induced by localized infection of muskmelons with Colletotrichum lagenarium. Plant Science Letters 28, 103-110 . 8 . HÄCHLER, H . & HOHL, H . R . (1984) . Temporal and spatial distribution patterns of collar and papillae wall appositions in resistant and susceptible tuber tissue of Solanum tuberosum infected by Phytophthora infestons. Physiological Plant Pathology 24, 107-118. 9 . HAMMERSCHMIDT, R. & Kuc, J . (1982) . Lignification as a mechanism for induced systemic resistance in cucumber . Physiological Plant Pathology 20, 61-71 . 10 . HAMMERSCHMIDT, R ., NUCKLES, E . M . & Kuc, J . (1982) . Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium . Physiological Plant Pathology 20, 73-82 . 11 . HAYASHI, T ., READ, S, M ., BUSSELL, J ., THELEN, M ., LIN, F.-C ., BROWN JR ., R . M . & DELMER, D . P . (1987), UDP-Glucose ; (1 - > 3)-ß-glucan synthases from mung bean and cotton . Plant Physiology 83, 1054-1062 . 12 . HINCH, J . M . & CLARKE, A . E . (1982) . Callose formation in Zea mays as a response to infection with Phytophthora cinnamomi . Physiological Plant Pathology 21, 113-124 . 13 . JENNS, A. E . & Kuc, J . (1980) . Characteristics of anthracnose resistance induced by localized infection of cucumber with tobacco necrosis virus . Physiological Plant Pathology 17, 81-91 . 14 . KAuss, H . & JEBLICK, W . (1985) . Activation by polyamines, polycations, and ruthenium red of the Ca"dependent glucan synthase from soybean cells . FEBS Letters 185, 226-230 . 15 . KAuss, H . (1987) . Callose-synthese . Regulation durch induzierten Ca'-Einstrom in Pflanzenzellen . .Naturwissenschaften 74, 275-281 . 16 . KAUSS, H . (1987) . Some aspects of calcium-dependent regulation in plant metabolism . Annual Review o! Plant Physiology 38, 47-72 . 17 . KAUSS, H . (1990) . Role of the plasma membrane in host-pathogen interactions . In The Plant Plasma Membrane - Structure, Function and Molecular Biology, Ed . by Ch . Larsson & 1 . M . Moller, pp . 320-350 . Springer Verlag, Berlin. 18 . KAUSS, H ., KOHLE, H . & JEBLICK, W . (1983) . Proteolytic activation and stimulation by Cas' of glucan synthase from soybean cells . FEBS Letters 158, 84-88 . 19 . LARSSON, CH ., KjELLBOM, P., WIDELL, S . & LUNDBORG, T . (1984) . Sidedness of plant plasma membrane vesicles purified by partitioning in aqueous two-phase systems . FEBS Letters 171, 271-276 . 20 . MAUCH, F ., MAUCH-MANI, B . & BOLLER, T. (1988) . Antifungal hydrolases in pea tissue . II . Inhibition of fungal growth by combinations of chitinase and ß-1,3-glucanase . Plant Physiology 88, 936-942 . 21 . MÉTRAUx, J . P . & BOLLER, TH . (1986) . Local systemic induction of chitinase in cucumber plants in response to viral, bacterial and fungal infections . Physiological and Molecular Plant Pathology 28, 161-169 .
228
I . Schmele and H . Kauss
22 . Rosy, D ., ToPPAN, A. & EsQUERRÉ-TUGAYÉ, M-T . (1988) . Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment . Physiological and Molecular Plant Pathology 33, 409-417 . 23 . SIEGRIST, J . & KAUSS, H . (1990) . Chitin deacetylase in cucumber leaves infected by Colletotrichum lagenarium . Physiological and Molecular Plant Pathology 36, 267-275 . 24 . STUMM, D . & GESSLER, C . (1986) . Role of papillae in the induced systemic resistance of cucumbers against Colletotrichum lagenarium . Physiological and Molecular Plant Pathology 29, 405-410 . 25 . WALDMANN, T ., JEBLICK, W . & KAUSS, H . (1988) . Induced net Ca"-uptake and callose biosynthesis in suspension cultured soybean cells . Planta 173, 88-95 . 26 . XUEI, X. L ., JÄRLFORS, U . & Kuc, J . (1987) . Ultrastructural changes associated with induced systemic resistance of cucumber to disease : host response and development of Colletotrichum lagenarium in systemically protected leaves . Canadian Journal of Botany 66, 1028-1038 .
221
Enhanced activity of the plasma membrane localized callose synthase in cucumber leaves with induced resistance 1 . SCHMELE and H. KAUSSt Fachbereich Biologie der Universität Kaiserslautern, Postfach 3049, D-6750 Kaiserslautern, F .R.G . (Accepted for publication July 1990)
The resistance of the first true leaf of cucumber to Colletotrichum lagenarium was increased by infecting the cotyledon or the tip of the first leaf with C . lagenarium or tobacco necrosis virus . Microsomes and plasma membranes, prepared from first leaves in which resistance was induced, exhibited increased specific activity of the Cat'-regulated 1,3-ß-glucan (callose) synthase . This enzyme activity is latent in epidermal cells of unchallenged healthy leaves but after activation renders the cells capable of a rapid production of callose-containing papillae . Activation occurs as soon as the Ca" permeability of the plasma membrane is perturbed by attempted fungal penetration . INTRODUCTION Systemically induced resistance in cucurbits against Colletotrichum lagenarium is associated with an increased ability of the host to prevent penetration of the epidermal cell wall [9, 13, 24] . This induced penetration might result, in part, from the ability of the cells to react by cell wall lignification at the site of attempted penetration [9] . The increased lignification in systemically resistant plants is reflected in cucumber at a biochemical level by enhanced peroxidase activity [10] and in muskmelon by increased hydroxycinnamate : CoA ligase activity [T] . It appears that induced resistance involves a developmentally regulated increased synthesis of enzymes that allows defence reactions to proceed more effectively when required . The systemic induction of chitinase [21] and 1,3-ß-glucanase [2] in cucumber and of chitinase in muskmelon [22] also involves enzyme synthesis . These lytic enzymes could operate by directly suppressing the growth of fungal hyphae in the course of penetration [20] . Consistent with this idea is the localization of chitinase in the apoplastic space of cucumber [3] . As an additional defence-mechanism, callose-containing papillae [2] are formed earlier and are more efficient in preventing penetration in cucumber leaves exhibiting induced resistance [24] . The activity of the plasma membrane-localized Ca"-dependent 1,3-ß-glucan synthase is latent in suspension cells but increases rapidly when local perturbations of the plasma membrane result in an influx of Ca" [15-17, 25] . The research reported in this paper shows that the activity of 1,3-,O-glucan f To whom correspondence should be addressed . 0885--5765/90/090221 +08 $03 .00/0
© 1990 Academic Press Limited I3-2
222
I . Schmele and H . Kauss
synthase is systemically enhanced in resistant' leaves and this enzyme might enable the plasma membrane to form papillae by more `rapidly depositing callose .
MATERIAL AND METHODS
Plant materials Cucumber plants (Cucumis sativus L . cv . Mervita) were grown as described [23] . Induction of systemic resistance was performed by placing six drops (10 µl) of a spore suspension (10 6 spores ml - ') of C. lagenarium on one cotyledon of plants in which the first true leaf had nearly expanded . Alternatively, both cotyledons were gently rubbed with a suspension of tobacco necrosis virus (TNV) containing 6 mg ml -1 Celite [23] . The plants were then allowed to grow for various times before systemic resistance was assessed by placing 20 drops (5 µl) of a C . lagenarium spore suspension (10 6 spores ml - ') on the first leaves .
Microsome preparations The first true leaves from four to seven plants (as indicated) were cut, and 0 . 5 g were homogenized at 0 °C [5, 23] in 5 ml of 100 mm Hepes/NaOH (pH 7 . 0) containing 10 mm EGTA and 5 mm dithiothreitol . The homogenate was centrifuged at 480 g for 5 min and the pellet (cell wall debris plus some adhering parts of plasma membranes) then resuspended in 5 ml of 50 mm Hepes/NaOH (pH 7 . 0) containing 1 mm dithiothreitol. Microsomes from the supernatant were pelleted at 48000 g for 5 min and the pellet washed twice in 5 ml of 50 mm Hepes/NaOH (pH 7 . 0) containing 1 mm dithiothreitol before resuspending in 5 ml of the same buffer .
Plasma membrane preparations Leaves (2 . 5 g) were homogenized in 10 ml of 100 mm Hepes/NaOH (pH 7 . 0) containing 5 mm dithiothreitol, 0.25 M sucrose and 10 mm EDTA . The homogenate was fractionated by centrifugation as described [5] and the microsome fraction suspended in 3 ml of 10 mm Hepes/NaOH (pH 7 . 0) containing 0 . 25 M sucrose and 1 mm EDTA . The microsome suspension (1 . 24 g, about 1 ml) was added to the same buffer (8. 15 g) containing 0 . 58 g polyethyleneglycol 4000 and 0 . 58 g dextran T500 after cooling to 0 ° C and mixed gently [19] . To allow separation of the total 3 ml of microsome fraction, three samples were run in parallel . They were centrifuged (1000 g for 3 min at 2 °C) and about 90% of each upper phase was recovered . A fresh upper phase was prepared by centrifugation of the same mixture as above but without microsomes, and this was added to each lower phase to give a second mixture and recentrifuged . The combined upper phases from each of the three samples were diluted 5-fold by volume with 10 mm Hepes/NaOH (pH 7 . 0) containing 1 mm EDTA and the membranes pelleted by centrifugation (100000 g, 1 h) . The resulting plasma membrane fraction was resuspended in I ml of 50 mm Hepes/NaOH (pH 7 . 0) containing 1 mm dithiothreitol .
Assay of 1,3-#-glucan synthase The enzyme assay [5] used 50 µl of the membrane suspension and 50 µi of assay buffer . In some experiments (Tables 2 and 3) the assay buffer consisted of 50 mm Tes/NaOH
223
Activity of callose synthase in cucumber TABLE I
Activity of the 1,3-ß-glucan synthase in various subcellular fractions derived from the first leaves of control and systemically resistant cucumber plants
Activity' in first leaves from Control Crude homogenate' After centrifugation at 480 g Supernatant Pellet Washed microsomes' Specific activity of microsomes' Specific activity [%] Lesions' Number Diameter [mm]
Resistant
53
63
61 15 30 11 100
76 19 43 18 163 3-5+0-7 1 . 6±0. 5
19-0+1-4 4. 6±0 . 1
'Total mU in the preparation derived from 0 . 5 g of leaves . °A single cotyledon on each of seven seedlings was inoculated with C. lagenarium and 7 days later the first leaves were harvested and 0 . 5 g homogenized in 5 ml of butler. Control plants were not inoculated . `Microsomes were twice pelleted and resuspended in buffer (5 ml per 0. 5 g of leaves) . '[mU mg- ' protein] . 'Resistance was assayed by challenge inoculating with 20 drops of C . lagenarium spores on the first leaves of two plants grown in parallel .
TABLE
2
Specific activity of microsomal 1,3-fl-glucan synthase in cucumber leaves infected at the tip with TNV to induce systemic resistance against C . lagenarium
Specific activity [mU mg - ' protein] Segment
Control
Resistant'
Increase [-fold]
1 (tip)° 2 3 4 (base)
5. 2 5.3 3.2 3.7
22. 0 15 . 1 16. 4 17. 0
4 .2 2.8 5.1 4 .6
'Induced systemic resistance was assayed by spreading 20 drops of C. lagenarium spores over the entire first leaf surface except for the tip, 4 days after inoculating the tip with TNV . 'The leaves from 4 plants were infected at their tip (about 1 . 5 cm) with TNV and cut after 4 days into four segments of equal width . The segments were homogenized and microsomes prepared as described in footnotes ° and a in Table 1 .
(pH 7 . 0) 16 % w/v glycerol, 20 mm cellobiose, 0 .04 % w/v digitonin, 10 mm MgCl,, 4 mm EGTA, 4 mm CaCl2' It contained a final free Ca" concentration of about 60 RM . In other experiments (Table 1) it consisted of 50 mm Hepes/NaOH (pH 7 . 0) with the same additions . As certain cell fractions assayed with this buffer contained some EDTA, the final concentration of free Ca" was between 0 . 1 and 1 . 0 mm, thus providing conditions in which the enzyme was saturated with Ca" [5, 15, 18] . The enzyme
224
I . Schmele and H . Kauss TABLE 3
Specific activity of 1,3-ß-glucan synthase in plasma membranes from the first leaves of control and systemically resistant cucumber plants
First leaves from Control Microsomes Total activity [mU]' Spec . activity [mU mg r protein] Plasma membranes° Total activity [mU] ° Spec . activity [mU mg' protein] Purification [-fold] Lesions' Number Diameter [mm]
30 .3 10 11 .7 140±29 14 18. 7±0. 5 5 . 1±0 . 5
Resistant'
78 .6 21 (2. 1)e 30 .4 293+21 (2. 1)c 13 .9 4 .0±1 . 6 1 .6±0. 4
'Systemic resistance was induced as described in Table 1 . 'Total activity in the preparation derived from 2 . 5 g leaf fresh weight . 'Increase over control [-fold] . ° Plasma membranes were prepared from microsomes in three PEG/dextran two phase systems run in parallel . The means ± SD of the specific activities from these three samples are given . 'Resistance was assayed by challenge inoculating the first leaves with 20 drops of C . lagenarium spores . The results are means+ SD on the first leaves from three plants .
reaction was started by the addition of 5 gl of radiolabelled UDP-glucose to give a final concentration 0.8 mm [5] . The reaction was terminated after 10 or 20 min at 25 ° C and the amount of product determined [5] . Protein was measured with Coumassie blue [5] using BSA as a standard . RESULTS 1,3-ß-glucan
synthase
activity in microsomes
All subcellular fractions from the true first leaves of plants exhibiting systemic resistance induced by C. lagenarium showed an increased 1,3-ß-glucan synthase activity over the uninduced controls (Table 1) . However, in both the control and resistant plants the total enzyme activity in the supernatants was higher than in the crude homogenates, although the resuspended pellets also exhibited some activity because of plasma membrane remains adhering to cell wall debris . The higher 1,3-ß-glucan synthase activity in the supernatants is most likely due to the fact that a significant fraction (about one third) of the crude homogenate volume is composed of bulky leaf constituents devoid of activity (e .g. cell walls and nuclei) and their removal as a pellet increases the proportion of microsomes in the samples assayed from the supernatants . The increased 1,3-#-glucan synthase activity was shown to be membrane-associated by measurement of the specific activity of the washed microsomes (Table 1) . In five repeat experiments similar to the one described in Table 1, the specific activity of 1,3ß-glucan synthase in the microsomes from resistant leaves was 157 ± 22 % of that in the control plants .
Activity of callose synthase in cucumber
225
The increase in specific activity was not specific to plants induced by C . lagenarium since in five similar experiments using TNV as the inducing agent, an increase in the specific activity of 1,3-fl-glucan synthase of 184±38% was observed 4 days after inoculation . However, no increase in 1,3-fl-glucan synthase was observed in the first leaves 7 days after inoculation of the cotyledons with TMV (data not shown), a pathogen that does not produce necrotic lesions nor induce systemic resistance in cucumber [13] . Inoculating the tip of the first true leaf with TNV induced systemic resistance in the rest of this leaf against C. lagenarium . The results of one experiment, given in Table 2, show that 17-5+2-3 lesions developed per leaf in the control uninduced plants while only 5 . 5 ± 2 . 1 lesions developed in TNV-induced leaves 7 days after challenge infection . The lesions on the control plants were also larger than on the induced resistant plants . The specific activity of 1,3-ß-glucan synthase in microsomes from an area of the leaf adjacent to the site of TNV-inoculation as well as from the entire leaf was considerably higher 4 days after inoculation than in controls (Table 2) . In eight similar repeat experiments the increase in 1,3-fl-glucan synthase at the tip was 2 . 6±0 . 7 fold greater than in the controls . A time course study revealed that the earliest detectable increase in 1,3-ß-glucan synthase occurred in the tip segments 2 days after inoculation and was fully expressed throughout the leaves 2 days later (data not shown) . 1,3-fl-glucan synthase activity in the plasma membrane
Partitioning the microsomes in a two phase PEG/dextran system, which selects for sealed rightside-out plasma membrane vesicles [5, 19], resulted in a similar enrichment of the 1,3-fl-glucan synthase activity in control and resistant plants with a two-fold higher specific activity in the plasma membranes from systemically resistant plants (Table 3) . Five repeat experiments showed a mean increase in the specific activity in the plasma membranes from resistant leaves of 183±25% . Some properties of 1,3-ß-glucan synthase from cucumber
1,3-fl-glucan synthase activity in microsomes from cucumber leaves was studied with regard to certain regulatory properties . The detailed results are not given because the properties were in general similar to those reported for other plant species . Thus results are presented here as a summary . The cucumber enzyme requires Ca" for activity, with a half-saturation below 1 µM free Ca", even in the presence of 5 mm Mg" . In the absence of Mg", Ca" activation was synergistically enhanced by 50 IAM spermine and additively by poly-L-ornithine (25 µg 125 .tl - ' assay) . Enzyme activity under standard assay conditions increased up to 10-fold after addition of digitonin (0 . 019 %, w/v, final conc .), presumably due to the fact that the 1,3-ß-glucan synthase was present in rightside-out plasma membrane vesicles and the digitonin increased the permeability of the vesicles and thus increased access for substrate and activators to the cytoplasmic membrane surface . The substrate saturation curve was sigmoidal between 10 and 100 gm UDP-glucose, indicating a complex regulatory behaviour . Therefore, about 0 . 8 mm UDP-glucose was required for the standard assay . The properties of the enzyme in microsome fractions of control and sytemically resistant leaves were similar, but the enzyme from induced resistant leaves exhibited higher activities at the same activator concentrations .
226
I . Schmele and H . Kauss
DISCUSSION The systemically induced resistance in cucumber to fungi is clearly associated with a decrease in penetration efficiency [9, 24, 26] . This reduced efficiency appears to result from a number of factors which result from mechanisms that are systemically programed during the pre-infection induction process and then become triggered by the fungal attack . Some fungal penetration attempts fail without any cytologically visible host reaction [24] . The presence of preformed fungitoxic substances such as chitinase [3] in the apoplast of induced plants may relate to this observation . Cytologically detectable reactions are involved including the incorporation of ligninlike material in the epidermal cell wall [9] and/or the deposition of papillae below the area of attempted penetration [24, 26] . The ability to react rapidly in this manner appears to result from a developmentally regulated induction of enhanced activities of enzymes, as previously shown for enzymes involved in phenylpropanoid production [7] and polymerization [10] . Papillae, in general, have been shown to contain callose among other substances [ref. 1, 8, 12, and literature cited there] . These cell wall appositions occur at very localized sites and their composition can only be evaluated by relatively non-specific histochemical methods . The contribution of callose deposition in papillae to resistance remains, therefore, controversial . Recently, careful time-course studies combined with the use of 2-deoxy-n-glucose to inhibit callose formation in the ml-o barley/powdery mildew system have provided evidence that callose deposition can contribute to resistance [1] . In cucumber, rapid formation of full-size papillae is associated with penetration inhibition in systemically resistant plants [2, 24] . Cucumber papillae are poorly characterized biochemically although electron-dense and -opaque layers have been observed in electron micrographs [26], possibly corresponding to lignin-like and polysaccharide material, respectively . The presence of callose in cucumber papillae has been shown by fluorescence with aniline blue [2] . The properties of the cucumber 1,3-fl-glucan synthae, namely its stimulation by Ca", Mg", digitonin, spermine or poly-t .-ornithine as well as its partitioning in the two phase system (Table 3), all show that a plasma membrane-located 1,3-#-glucan synthease, formerly called glucan synthase II . This enzyme has been implicated in callose synthesis [5, 11, 14-16, 18] . Several unsuccessful attempts to purify this enzyme have been made [4, 6] . However, these attempts have provided indirect evidence that the enzyme has a complex structure and probably consists of several subunits exhibiting different functions . Thus, no conclusions about whether the increased specific activity measured under standard assay conditions in microsomes and plasma membranes from systemically resistant leaves is due to an overall increase in all enzyme components or only relate to the regulatory Ca"-binding subunits can be drawn . The observation that the 1,3-#-glucan synthase activity in the plasma membranes of cucumber leaves with systemically induced resistance is enhanced, provides further evidence for the general notion that defence-related enzymes become more active due to long-term developmental processes . In the case of callose formation, allosteric Ca" activation is a major parameter of short-term regulation of 1,3-#-glucan synthase, although additional signals are likely to be required [16, 17] . This activation mechanism
227 Activity of callose synthase in cucumber for latent 1,3-#-glucan synthase, which occurs with higher activity in cucumber leaves exhibiting increased resistance, is consistent with the cytological observation that in these leaves a higher proportion of full-sized papillae is formed below the site of fungal attack [24] . How callose formation co-operates with the other processes which contribute to papillae formation remains to be established . This work was financially supported by the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie .
REFERENCES 1 . BAYLES, C . J ., GHEMAWAT, M . S., AIST, J . R . (1990) . Inhibition by 2-deoxy-D-glucose of callose formation, papillae deposition, and resistance to powdery mildew in an ml-o barley mutant . Physiological and Molecular Plant Pathology 36, 63-72 . 2 . BINDER, A., BAER, G ., HOFMANN, C. & KOVATS, K . (1989) . Mechanisms in systemic induced disease resistance. In Signal Molecules in Plants and Plant-Microbe Interactions, Ed . by B, J . J . Lugtenberg, pp . 267-272 . Springer Verlag, Berlin . 3 . BOLLER, Ta. & MÉTRAUX, J . P . (1988) . Extracellular localization of chitinase in cucumber . Physiological and Molecular Plant Pathology 33, 11-16 . 4 . DELMER, D . P . (1987) . Cellulose biosynthesis . Annual Review of Plant Physiology 38, 259-290 . 5 . FINK, J ., JEBLICK, W ., BLASCHEK, W . & KAUSS, H . (1987) . Calcium ions and polyamines activate the plasma membrane-located 1,3-#-glucan synthase. Planta 171, 130-135 . 6 . FINK, J ., JEBLICK, W. & KAUSS, H . (1990) . Partial purification and immunological characterization of 1,3-fl-glucan synthase from suspension cells of Glycine max. Planta 181, 343-348 . 7 . GRAND, C . & ROSSIGNOL, M . (1982/83) . Changes in the lignification process induced by localized infection of muskmelons with Colletotrichum lagenarium. Plant Science Letters 28, 103-110 . 8 . HÄCHLER, H . & HOHL, H . R . (1984) . Temporal and spatial distribution patterns of collar and papillae wall appositions in resistant and susceptible tuber tissue of Solanum tuberosum infected by Phytophthora infestons. Physiological Plant Pathology 24, 107-118. 9 . HAMMERSCHMIDT, R. & Kuc, J . (1982) . Lignification as a mechanism for induced systemic resistance in cucumber . Physiological Plant Pathology 20, 61-71 . 10 . HAMMERSCHMIDT, R ., NUCKLES, E . M . & Kuc, J . (1982) . Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium . Physiological Plant Pathology 20, 73-82 . 11 . HAYASHI, T ., READ, S, M ., BUSSELL, J ., THELEN, M ., LIN, F.-C ., BROWN JR ., R . M . & DELMER, D . P . (1987), UDP-Glucose ; (1 - > 3)-ß-glucan synthases from mung bean and cotton . Plant Physiology 83, 1054-1062 . 12 . HINCH, J . M . & CLARKE, A . E . (1982) . Callose formation in Zea mays as a response to infection with Phytophthora cinnamomi . Physiological Plant Pathology 21, 113-124 . 13 . JENNS, A. E . & Kuc, J . (1980) . Characteristics of anthracnose resistance induced by localized infection of cucumber with tobacco necrosis virus . Physiological Plant Pathology 17, 81-91 . 14 . KAuss, H . & JEBLICK, W . (1985) . Activation by polyamines, polycations, and ruthenium red of the Ca"dependent glucan synthase from soybean cells . FEBS Letters 185, 226-230 . 15 . KAuss, H . (1987) . Callose-synthese . Regulation durch induzierten Ca'-Einstrom in Pflanzenzellen . .Naturwissenschaften 74, 275-281 . 16 . KAUSS, H . (1987) . Some aspects of calcium-dependent regulation in plant metabolism . Annual Review o! Plant Physiology 38, 47-72 . 17 . KAUSS, H . (1990) . Role of the plasma membrane in host-pathogen interactions . In The Plant Plasma Membrane - Structure, Function and Molecular Biology, Ed . by Ch . Larsson & 1 . M . Moller, pp . 320-350 . Springer Verlag, Berlin. 18 . KAUSS, H ., KOHLE, H . & JEBLICK, W . (1983) . Proteolytic activation and stimulation by Cas' of glucan synthase from soybean cells . FEBS Letters 158, 84-88 . 19 . LARSSON, CH ., KjELLBOM, P., WIDELL, S . & LUNDBORG, T . (1984) . Sidedness of plant plasma membrane vesicles purified by partitioning in aqueous two-phase systems . FEBS Letters 171, 271-276 . 20 . MAUCH, F ., MAUCH-MANI, B . & BOLLER, T. (1988) . Antifungal hydrolases in pea tissue . II . Inhibition of fungal growth by combinations of chitinase and ß-1,3-glucanase . Plant Physiology 88, 936-942 . 21 . MÉTRAUx, J . P . & BOLLER, TH . (1986) . Local systemic induction of chitinase in cucumber plants in response to viral, bacterial and fungal infections . Physiological and Molecular Plant Pathology 28, 161-169 .
228
I . Schmele and H . Kauss
22 . Rosy, D ., ToPPAN, A. & EsQUERRÉ-TUGAYÉ, M-T . (1988) . Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment . Physiological and Molecular Plant Pathology 33, 409-417 . 23 . SIEGRIST, J . & KAUSS, H . (1990) . Chitin deacetylase in cucumber leaves infected by Colletotrichum lagenarium . Physiological and Molecular Plant Pathology 36, 267-275 . 24 . STUMM, D . & GESSLER, C . (1986) . Role of papillae in the induced systemic resistance of cucumbers against Colletotrichum lagenarium . Physiological and Molecular Plant Pathology 29, 405-410 . 25 . WALDMANN, T ., JEBLICK, W . & KAUSS, H . (1988) . Induced net Ca"-uptake and callose biosynthesis in suspension cultured soybean cells . Planta 173, 88-95 . 26 . XUEI, X. L ., JÄRLFORS, U . & Kuc, J . (1987) . Ultrastructural changes associated with induced systemic resistance of cucumber to disease : host response and development of Colletotrichum lagenarium in systemically protected leaves . Canadian Journal of Botany 66, 1028-1038 .